Tumor necrosis factor- alpha (TNF) is now known to be a critical component of the inflammatory process and excess TNF levels plays a pathological role in severe inflammatory diseases including rheumatoid arthritis. Therefore a greater understanding of how TNF receptor 1 (TNFR1) signaling is regulated may suggest new targets for therapeutic interventions. Ligation of TNFR1 leads either to the activation of the NF-kappaB pathway or to the activation of the apoptosis pathway. In another NIAID-funded grant, we proposed to study the mechanism by which TNFR1 signaling to the apoptosis pathway can be shut down by the NF-kappaB pathway, focusing on that mediated by A20. In addition, one of our aims was to identify other novel genes that can inhibit TNF signaling to the apoptosis pathway. In Aim 1 of this application, we propose to develop an expression cloning strategy to functionally isolate cDNAs that inhibit TNFR1 signaling to apoptosis. This strategy will be based on using a Jurkat T cell mutant we described previously in our grant that is deficient in IKK activity and thus highly sensitive to TNF-mediated apoptosis. The presence of a cDNA encoding a protein that inhibits the apoptosis pathway in these cells will confer a survival advantage to TNF-treated cells and can be cloned from the surviving cells. In addition to molecules that inhibit the apoptosis pathway, the identification of molecules that inhibit the NF-kappaB pathway would also advance our understanding of TNF signaling. In order to do so in Aim 2, we have generated a different Jurkat cell line that undergoes suicide whenever NF-kappaB is activated. Therefore, this cell line can be used as a platform to screen for genes that inhibit the NF-kappaB pathway, again using death as a selection tool. Cells harboring cDNAs that inhibit the NF-kappaB pathway will survive TNF or PMA treatment and the cDNA can then be isolated. Both cloning strategies are based on similar principles and required similar methodologies. The development of these approaches will accelerate the identification of genes with specific regulatory functions in TNF signaling.